The long-term objectives of this program are to define the transport mechanisms for lipids and lipid precursors into cells and organelles and to elucidate relationships between transport reactions and metabolism of lipids. The work in this proposal focuses on two microchondrial enzymes, electron transfer flavoprotein (ETF) and electron transfer flavoprotein dehydrogenase (ETF-DH), a membrane-bound, iron-sulfur flavoprotein. These proteins comprise part of an electron transfer system between eight soluble mitochondrial flavoprotein dehydrogenases and the respiratory chain. The specific aims of the work are to determine the role of ubiquionone (Q10) in electron transfer between ETF-DH and mitochondrial Complex III and characterize the reaction of ETF-DH with Q10 and short chain Q analogs. These experiments will employ Q-depleted submitochondrial particles and ETF-DH reconstituted into phospholipid vesicles with Q10 and Complex III. The reconstitution experiments will permit detailed investigation of the enzyme in a membrane environment and determination of the function of phospholipid in the activity of the ETF-DH. The function of the redox centers of ETF-DH will be investigated using a modified ETF-DH from which the Fe4-S4 cluster has been removed. An important objective of the proposed work is to determine whether deficiency of ETF or ETF-DH result in the inherited metabolic disease, glutaic acidemia type II (GA2). ETF and ETF-DH will be studied in fibroblasts from patients with GA2. The biochemical pattern of the disease indicates a defect in electron transfer between ETF-linked dehydrogenases and the respiratory chain. The genetic diversity of the patients suggests that more than one protein may be defective, such that ETF may be defective in some patients and ETF-DH may be defective in others. The presence of the proteins will be determined by Western blotting of fibroblast mitochondrial proteins. The synthesis, translocation into mitochondria and primary structures of the proteins will be investigated in at least three GA2 fibroblast lines to define the molecular bases of the disease.